PIN1 inhibitor API-1

Reduction of nitric oxide and DNA/RNA oxidation products are associated with active disease in systemic lupus erythematosus patients

The aims of the present study were to evaluate biomarkers of oxidative and nitrosative stress in systemic lupus erythematosus (SLE) patients, in particular products of DNA/RNA oxida- tive damage and their correlation with disease activity. This study included 188 controls and 203 patients; 153 with inactive SLE (SLEDAI < 6) and 50 with active SLE (SLEDAI ≥ 6) without renal impairment. Oxidative stress was assessed by tert-butyl hydroperoxide—ini- tiated by chemiluminescence, advanced oxidation protein products (AOPP), total radical- trapping antioxidant parameter (TRAP), nitric oxide metabolites (NOx), and DNA/RNA oxidation products. Patients with SLE showed increased oxidative stress, as demonstrated by the augmentation of lipid hydroperoxides (p < 0.0001) and AOPP (p < 0.001) and reduced total antioxidant capacity (p < 0.0001), without differences between patients with active dis- ease and in remission. NOx levels and DNA/RNA oxidation products were inversely and independently associated with disease activity (p < 0.0001 and p = 0.021, respectively), regard- less of BMI and prednisone use. The linear regression analysis showed that about 5% of the SLEDAI score can be explained by the levels of DNA/RNA oxidation products (r2:0.051; p = 0.002) and about 9% of this score by the levels of NOx (r2:0.091; p < 0.0001). This study provides evidence for an inverse association between serum NOx levels and DNA/RNA oxi- dation products and SLE disease activity, suggesting that oxidative/nitrosative stress markers may be useful in evaluating SLE disease activity and progression of the disease. Lupus (2017) 0, 1–6. Key words: Systemic lupus erythematosus; oxidative stress; nitric oxide; DNA oxidation; RNA oxidation; SLEDAI Introduction Systemic lupus erythematosus (SLE) is an auto- immune syndrome characterized by autoantibody production, especially against nuclear components. It has been suggested that the increased production of reactive oxygen and nitrogen species (ROS and RNS, respectively), as an important arm of the innate immune response, may favor its development.1 The role of ROS/RNS in the deregulation of apoptosis and the delay in clearance of apoptotic cells may generate neo-epitopes that subsequently stimulate broad spectrum of autoantibody forma- tion, leading to inflammation and organ damage in SLE.Oxidative stress and pro-inflammatory cytokines may stimulate NO production by an increase in inducible nitric oxide synthase (iNOS) expression. Nevertheless, analysis of the concentration of nitric oxide metabolites (NOx) in patients with SLE has shown contradictory results; some authors have not found any alteration,3 whereas others reported structure, thus favoring their recognition by the immune system and consequent formation of anti-double-stranded DNA (anti-dsDNA).5 Despite the importance of nucleic acid oxidation in the induction of autoantibody production, few studies have evaluated this marker in patients with SLE and investigated its association with disease activity.Therefore, the aim of this study was to evaluate biomarkers of oxidative and nitrosative stress in SLE patients, in particular products of DNA/ RNA oxidative damage, and their correlation with disease activity. Subjects and methods The study included 391 subjects; 188 healthy indi- viduals (control group) were selected from among blood donors and 203 patients with SLE were selected from the ambulatory of Rheumatology of the University Hospital of Londrina, Parana´ , Brazil. SLE was diagnosed using the American College of Rheumatology (ACR) 1997 revised cri- teria. Patients with SLE were also divided in two groups: patients with active (n = 50) or inactive dis- ease (n = 153). A Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score ≥6 was used to classify SLE as active.6 Information on lifestyle factors and medical history were obtained at clinical evaluation. Disease duration, organ involvement, and therapy were rec- orded for each patient. No patient with SLE pre- sented proteinuria. The individuals of both groups did not drink alcohol regularly. None of the par- ticipants in the study presented heart, thyroid, renal, hepatic, gastrointestinal, or oncological dis- ease, and none were receiving estrogen replacement therapy or antioxidant supplements. This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and the Ethical Committee of the University of Londrina, Parana´ , Brazil, approved all procedures involving human subjects (approval number 210.328). Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. Serum comple- ment factors C3 and C4 levels were measured using an immunoturbidimetric assay (C8000 Architect Abbott Laboratories, Abbott Park, IL, USA). Antinuclear antibodies (ANA) were quantified using indirect immunofluorescence with HEp2 cells as substrate (IFI-ANA-HEp 2-IgG, Viro- Immun-Labor-Diagnostika, GmbH, Oberursel, Germany) and were considered significant when titers ≥1:160. Antibodies against double-stranded DNA (anti-dsDNA) were quantified using enzyme linked immunoassay (ELISA, anti-dsDNA, Orgentec Diagnostika, GmbH, Germany) and were considered significant when titers ≥20 IU/mL. Peripheral blood samples were obtained with ethylenediamine tetraacetic acid (EDTA), as anti- coagulant and antioxidant, for evaluating oxidative stress. All samples were centrifuged at 3000 r/min for 15 minutes, and plasma aliquots were stored at a freezer temperature of –70 ºC until use. Lipid hydro- peroxides in plasma was evaluated by Tert-butyl hydroperoxide-initiated chemiluminescence (CL- LOOH). Advanced oxidation protein products (AOPP) were determined in the plasma samples using the semi-automated method described by Witko-Sarsat et al.7 The measurement of DNA/ RNA oxidative damage products (DNA/RNA oxi) was performed by ELISA (Cayman Chemical Company, Ann Arbor, MI, USA) and detected all three oxidized guanine species;8-hydroxy-2r- deoxyguanosine from DNA, 8-hydroxyguanosine from RNA, and 8-hydroxyguanine from either DNA or RNA. Serum levels of nitric oxide metab- olites (NOx) were assessed by nitrite (NO —) and nitrate (NO3–) concentration, according to the Griess reaction, supplemented by the reduction of nitrate to nitrite with cadmium. Total Radical- Trapping Antioxidant Parameter (TRAP) was determined as reported previously by Repetto et al.8 The system was calibrated with the vitamin E analog Trolox. Statistical analysis Categorical data were analyzed by Fisher’s exact test or 2 test when appropriate. The results were demonstrated through absolute number. Comparison between control group and SLE patients, SLE group with active and inactive dis- ease, and positive and negative anti-dsDNA were made using the Mann–Whitney test, with data expressed as median (25%–75%). The results of these univariate statistical analyses were used to delineate the significant explanatory variables to be used, as determinants of independent association with diagnostic groups, in a subsequent logistic regression analyses. Bivariate logistic regression analysis was used to define the significant associ- ation of SLE versus controls, and active versus inactive disease using the biomarkers, with p < 0.10. Linear regression was applied between SLEDAI and NOx or DNA/RNA oxidative damage. All tests were two-tailed and a p-value of 0.05 was used for statistical significance. All ana- lyses were conducted with SPSS 20.0 software (SPSS, Chicago, IL, USA). Results The study included 203 patients with SLE, with an average duration of disease of 9 years (4.0–14.0 years). 71.4% presented with a positive ANA, 51% with a positive anti-dsDNA (≥20 IU/mL), and 25% with active disease (SLEDAI ≥ 6). In rela- tion to the therapy, 97% of SLE patients were using corticosteroids (prednisone average of 7.5 mg/day), 71% antimalarials, and 41% other immunosuppressors.The SLE patients group had significantly more non-Caucasian subjects than the control group (p = 0.016), and SLE patients were older (p = 0.006) and had higher BMI (p < 0.0001) than controls (Table 1). After adjusting for ethnicity, age, and BMI, SLE patients showed increased hydroperoxides (p < 0.0001) and AOPP (p < 0.001) levels. There was no significant change in serum oxidized DNA/RNA levels (p = 0.481). The serum levels of NOx (p = 0.017) and TRAP (p < 0.0001) were significantly reduced in SLE patients com- pared with the control group (Table 1). In a subsequent logistic regression analyses (data not shown), with the SLE group as dependent vari- able and the normal control group as the reference group and the significant biomarkers shown as explanatory variables, we found that hydroperoxides (p = 0.004), AOPP (p = 0.030), and BMI (p = 0.003) were significantly and positively associated with SLE, whereas TRAP (p < 0.0001) was significantly and inversely associated with SLE. However, age (p = 0.936) and ethnicity (p = 0.204) did not change the association between the biomarkers and SLE. There was no difference in NOx levels in relation to the control group (p = 0.595). Table 2 shows demographic, clinical, and oxidative stress biomarkers from SLE patients with inactive and active disease. There was no difference between the groups related to ethnicity (p = 0.198) and age (p = 0.366); however, SLE patients with active disease presented a higher frequency of women compared to those with inactive disease (p = 0.033). There was a trend of patients with active disease to present a higher BMI (p = 0.066) and to take higher doses of daily prednisone (p = 0.067). SLE patients with active disease had higher serum levels of anti-dsDNA (p = 0.018), and decreased C3 (p = 0.006) and C4 (p = 0.007) levels. The p-value was adjusted for sex, BMI, and prednisone dose, factors which may influ- ence oxidative stress parameters. Patients with active disease showed reduced serum levels of oxidized DNA/RNA (p = 0.003) and NOx levels (p < 0.0001) compared to those with inactive disease. There was no difference between the groups in relation to hydro- peroxides (p = 0.165), AOPP (p = 0.123), and TRAP (p = 0.869). The outcome of the logistic regression analysis with the active SLE group (SLEDAI ≥ 6) as dependent variable and the inactive SLE group (SLEDAI < 6) as the reference group and the sig- nificant biomarkers shown as explanatory variables was performed. We found that serum levels of oxi- dized DNA/RNA (p = 0.021) and NOx (p < 0.0001) were inversely associated with disease activity inde- pendently of the BMI and the prednisone dose (data not shown). Patients with SLE who showed positive anti- dsDNA had lower DNA/RNA oxidation products concentrations than those without anti-dsDNA (p = 0.0350, data not shown). Linear regression analysis demonstrated that the DNA/RNA oxida- tion products (r2:0.051; p = 0.002) and NOx levels (r2:0.091; p < 0.0001) account for 5% and 9% of the SLEDAI score, respectively (Figure 1). Discussion The main finding of this study was that the reduced levels of NOx and DNA/RNA oxidation products were inversely associated with SLE activity, regard- less of the corticosteroid use. In this study, patients with SLE showed increased oxidative stress, as demonstrated by the increase in hydroperoxides and AOPP and reduced total anti- oxidant capacity, and these biomarkers were inde- pendently associated with SLE, regardless of gender, age, and BMI. These data are consistent with a pre- vious study from our group9 and with studies from other researchers, which also demonstrated a redox imbalance in patients with SLE.10 However, in the present study, similarly to another study from our group,9 serum hydroperoxides, AOPP, and TRAP levels did not differ between SLE patients with active disease and those in remission. In that study, SLE patients with disease activity showed a significant and inverse correlation of daily prednisone doses with CL-LOOH or TRAP, which could explain the absence of significant difference in oxidative stress between active and inactive disease. It has been suggested that NO production via iNOS plays an important role in the pathogenesis of several systemic autoimmune disorders.10 In SLE, data are contradictory. Although some studies demonstrated higher serum NOx levels in SLE patients and association with SLEDAI score, anti- dsDNA, low complement, and lupus nephritis activ- ity,11 others did not find correlation between NOx and disease activity.9,3 In this study, we found that NOx levels were inversely and independently asso- ciated with disease activity evaluated by SLEDAI score. It is feasible to suggest that in conditions of inflammation and increased oxidative stress, NO is consumed in a reaction with superoxide anion, yield- ing the strong oxidant peroxynitrite, which in turn accelerates the lipid peroxidation reaction and decreases NO bioavailability.12 Therefore, although this study did not show significant changes in bio- markers of oxidative stress in patients with active disease, the reduction of NOx is a robust indication that this may be occurring. In the present study, DNA/RNA oxidation products were inversely and independently asso- ciated with disease activity. The most commonly used marker of oxidatively modified DNA is 8- hydroxy-2r-deoxyguanosine (8-OHdG), a product of oxidatively modified DNA base guanine. Lee et al.13 showed that increased plasma 8-OHdG levels correlated with more severe clinical symptoms and higher SLEDAI. On the other hand, Lunec et al.14 had shown no elevation of 8-0HDG urine excretion with inflammatory activity, but an accu- mulation of the altered DNA base in circulating immune complexes, resulting in a urine output l.000-fold lower than in normal controls. We are not aware of any study to date that evaluated DNA/RNA oxidative damage through the detection of all three oxidized guanine species in SLE patients. The reduction in DNA/RNA oxidation in the active SLE patients compared to inactive patients could be explained by increased binding of anti-DNA anti- bodies to modified DNA. Furthermore, ROS modi- fication of DNA can render these macromolecules more susceptible to interacting with circulating autoantibodies, thus promoting immune complex formation,15 and thus preventing detection of these molecules in serum. Some limitations must be considered in the present study. First, the cross-sectional design does not allow for inference causality. Second, several stu- dies have shown that sex, age, ethnicity, and cor- ticosteroid use influence oxidative stress and thus may be considered important confounding factors;however, these factors were controlled in this study. Third, another possible factor, which could inter- fere with NOx analysis, is the increase in BMI in active SLE patients. It was previously shown that serum NOx levels are inversely correlated with BMI and waist circumference.12 However, our data show that the reduction in NOx levels was associated with disease activity independently of BMI and prednisone dose. Conclusion The results of this study showed a strong inverse association between serum NOx levels and DNA/ RNA oxidation products and active SLE, suggest- ing that oxidative/nitrosative stress may be useful markers in SLE disease activity. In addition, our data showed the importance of measuring oxidative stress with several methodologies with different assumptions. The understanding of the complex redox mechanisms involved in the context PIN1 inhibitor API-1 of the disease will enable a more accurate view of the phenomena.